This invention relates to compositions and methods for monitoring changes in the local environment inside or outside of cells by detecting optical changes in an environment-sensitive reporter molecule. In particular, this invention relates to compositions which may be specifically incorporated into cells, or may be caused to be produced by them, which produce an optically detectable signal in response to a change in the environment in which the optically sensitive reporter molecule is present. This invention is particularly applicable to detecting the release of substances stored in exocytotic vesicles of cells, such as synaptic vesicles, wherein the compositions of the invention are localized in the vesicles and become exposed to the extracellular space upon release of the contents of the vesicles.
Various methods are known for monitoring molecular changes in local environments at a microscopic level. Detection of changes in the microenvironments involving active cells presents a particular challenge because many methods involve destruction or damage to the cells being analyzed. Although significant information has been obtained using electrophysiological techniques, these techniques have limited application for studying complex real time cell physiology and also may damage and thereby alter the cells being recorded. Also, electrophysiology is limited to detecting events involving a change in electrical potential.
Many cell process involve changes in the molecular microenvironment. Such processes include, for example, exocytosis and endocytosis which involves contact between the intracellular and extracellular environments, and changes in ion concentration associated with electrically active cells such as neurons and muscle cells. The release of cellular substances, in particular, is a generalized phenomenon of several cell types which is fundamental to the function of multicellular organisms. Blood cells, endocrine cells and neurons are examples of cell types particularly dependent on such processes. Blood granulocytes, for example, release various mediators of inflammation through exocytosis; endocrine cells release hormones required by other cells; and neurons release neurotransmitters packaged in synaptic vesicles. The ability to detect changes in the microenvironment caused, for example, by the fusion of an exocytotic vesicle with a plasma membrane and contact of the lumenal surface and contents of the vesicle with the extracellular space would provide information useful for understanding and modulating processes which causes release of such cellular substances.
Exocytotic events are particularly important to the proper function of neurons since synaptic transmission is dependent upon the controlled release of synaptic vesicles. Many problems in neurophysiology can be reduced to questions about the location, timing, and magnitude of synaptic activity, including, for instance, the integration of inputs by a single neuron, synaptic plasticity, and pattern classification and storage by neural networks. The study of these and related problems would greatly benefit from a method that allows direct recording from many synapses simultaneously, with the capacity to reliably detect single exocytotic events. Such a method would appear optimal because, on the one hand, central synapses generally transmit information via the fusion of a single synaptic vesicle (1,2), while, on the other hand, the computational power of the nervous system arises from networks containing large numbers of synapses (refs. 3-5).
While current electrophysiological methods allow the activity of individual synapses to be recorded, they do not permit populations to be studied. There is a practical limit to the number of cells that can be impaled simultaneously with intracellular electrodes, and importantly, an invasive method requires an a priori decision on which cells to study, making discovery difficult. Extracellular field recordings with multiple electrodes avoids some of these problems and thereby allows the collective activities of many cells to be measured, but does not permit activity to be ascribed to individual synapses or neurons (6,7). Optical imaging of light emission from fluorescent indicators of membrane potential or intracellular Ca2+ concentration (7-10) greatly increases spatial resolution but again, does not measure synaptic activity directly. An alternative optical approach that offers a direct gauge of synaptic activity is to load synaptic vesicles with fluorescent dyes and to observe dye release (11,12). However, this method is intrinsically incapable of resolving individual quanta, which can cause only a small decrease in total fluorescence. Despite their limitations, these techniques have opened a window on multicellular phenomena as diverse as the representation of visual scenes by retinal ganglion cells (13) and the emergence of cortical circuits during development (14). Methods that reveal the detailed patterns of synaptic inputs and outputs in entire networks can thus be expected to disclose important new physiological concepts operative at the relatively unexplored interface between cellular and systems neurophysiology.
Due to the importance to physiology of proper exocytotic processes in neurons and other cells types, it is therefore desirable to develop sensitive compositions and methods which can detect changes in the microenvironment inside or outside of cells including quantal exocytotic events in real time. Molecules which can detect changes in microenvironments would be useful as probes of cellular events involving changes in such microenvironments due to movement of molecules in solution or the spacial location of molecules associated with cell membranes. It would be particularly desirable to have available molecules that provided an optical signal upon encountering such a change in the microenvironment.
Various types of molecules have been used in the art for the detection of the presence of other molecular entities. Radiochemical labels have high sensitivity but are hazardous and must be used with appropriate caution. In addition, these labels are not useful for real time localization. Optical labels such as fluorescent molecules or other forms of dyes have also been coupled to molecules to act as reporters for the detection of specific molecular entities. Typically a reporter cable of generating an optical signal is bound to a specific binding molecule which is a member of a ligand binding pair. Such binding molecules are usually antibodies, specific binding proteins, e.g. receptors or peptide hormones which specifically binds a corresponding target ligand. These reporter-ligands are reagents which must be added from the external environment to the system under investigation. Their usefulness is therefore limited by their accessibility to the appropriate molecular target, nonspecific binding or diffusion to inappropriate locations and availability of appropriate binding pairs. Another type of molecular reporter comprises signal generating molecules expressed endogenously by a cell. Several bioluminescent proteins have been reported as useful as detectable labels for optically reporting the presence of a molecular entity.
The green fluorescent protein (GFP) of Aequora victoria, for example, is a naturally fluorescent protein with a p-hydroxybenzylideneimidazolone chromophore, created by in vivo cyclization and oxidation of the sequence Ser-Tyr-Gly (positions 65-67). The chromophore""s phenolic group, derived from Tyr-66, exists in two states of protonation, which in all likelihood underlie the protein""s two main excitation peaks at 395 and 475 nm (ref. 47). Several reports have characterized various bioluminescent proteins. See, for example, Cormier et al., xe2x80x9cRecombinant DNA Vectors Capable of Expressing Apoaequorinxe2x80x9d, U.S. Pat. No. 5,422,266; Prasher, xe2x80x9cModified Apoaequorin Having Increased Bioluminescent Activityxe2x80x9d, U.S. Pat. No. 5,541,309; Cormier et al., xe2x80x9cIsolated Renilla Luciferase And Method Of Use Thereofxe2x80x9d, U.S. Pat. No. 5,418,155; McElroy et al., xe2x80x9cRecombinant Expression of Coleoptera Luciferasexe2x80x9d, U.S. Pat. No. 5,583,024. The use of bioluminescent fusion proteins as reporters of gene expression has also been reported. See, for example, Harpold et al., xe2x80x9cAssay Methods And Compositions For Detecting And Evaluating The Intracellular Transduction Of An Extracellular Signalxe2x80x9d, U.S. Pat. No. 5,436,128; Tsein et al., xe2x80x9cModified Green Fluorescent Proteinsxe2x80x9d. International Application WO 96/23810; Gustafson et al., xe2x80x9cFusion Reporter Gene For Bacterial Luciferasexe2x80x9d, U.S. Pat. No. 5,196,524; and Chalfie et al. xe2x80x9cUses Of Green-Fluorescent Proteinxe2x80x9d, U.S. Pat. No. 5,491,084. Although GFP has been reported as a useful reporter molecule, its utility would be further enhanced if it could be made sensitive to changes in the microenvironment.
This invention provides compositions and methods useful for detecting changes in microenvironments. Many dynamic systems are dependent on compartmentalization of molecules with subsequent merging of compartments or release of the contents of one compartment into another compartment. Endocytosis and exocytosis are examples of biologic systems which involve compartmentalization of molecular entities. The compositions and methods of this invention are especially useful for detecting changes in the microenvironment associated with changes in compartmentalization. Detection of quantalcellular exocytotic events in real time such as those occurring in connection with the release of synaptic vesicles is made possible by this invention.
The method of this invention comprises detecting a change in the light emitting properties of a hybrid molecular reporter present in a first compartment upon contact with a second compartment. The hybrid molecular reporter comprises a targeting region and a reporter region which participates in a light-generating reaction upon contact with the second compartment. As applied to cells, the method of this invention enables the quantal detection of the release of components of exocytotic vesicles, especially synaptic vesicles. The method of this invention is anticipated to be applicable to detecting the release of vesicular contents within a cell as well.
This invention also provides hybrid molecular reporter molecules. At least two types of hybrid molecules are provided by this invention. One type of molecule is derived externally from the compartment in which it is to be introduced. When used to detect cellular processes, such hybrid molecules are typically not genetically encoded by the cells to which they are directed. In addition, the light-generating component provides a detectable optical signal which is environment sensitive. The other type of hybrid molecule provided by this invention is genetically encoded. These molecules themselves are of two types: a) those which include a targeting region and a reporter region wherein the reporter region is co-expressed with the targeting region and is capable of generating an optically detectable signal; and b) those which include a targeting region and a binding region which binds to a separate reporter molecule capable of generating an optically detectable signal but which is not encoded by the cell encoding the targeting-binding region and which is provided from the external environment.
The hybrid molecules of this invention are typically, but not necessarily, polypeptides since polypeptides are particularly well suited as targeting entities and may be genetically encoded and expressed. The compositions of this invention may be targeted to various types of multicompartment systems. In one embodiment, the hybrid molecules may be targeted to liposomes and used to monitor delivery to cells of substances, such as drugs contained by the liposomes. In another embodiment, the compositions of this invention may be targeted to intracellular locations, such as exocytotic vesicles, where they are not in contact with the extracellular environment until an exocytotic event occurs. Upon exocytosis, the interior of the exocytotic vesicle, including for example the lumenal side of the vesicle membrane, comes in contact with the extracellular environment. By coming into contact with the extracellular environment the compositions of this invention targeted to the vesicle cause a release of photons which may be detected as an optical event indicative of quantal exocytotic release.
The targeting polypeptide of the invention preferably is targeted to exocytotic vesicle membranes and the amino acid sequence required for generation of the optical signal is preferably located at the lumenal surface of the vesicle membranes. This embodiment encompasses molecules which generate an optical signal that directly reports neurotransmitter release. The detection of individual vesicle fusion events is made possible by this invention which also provides for the regeneration of probes for many rounds of recording. Preferred probes for this and other embodiments are genetically encoded proteins.
Genetic control of the expression of the probes of this invention allows recordings to be obtained from cells, cultures, tissue slices or exposed tissues of transgenic animals, and affords means to detect individual cells including neurons (by localized DNA transfer techniques), types of neurons (by cell-type specific promoters), or elements of a circuit (by recombinant viral vectors that spread through synaptic contacts). Such probes are useful for detecting the release of synaptic vesicle.
In one embodiment of this invention, hybrid reporter molecules which comprise a luciferase enzyme and at least a portion of a vesicle membrane protein are provided. These hybrid reporter molecules are referred to as xe2x80x9csynaptolucinsxe2x80x9d.
This invention also includes mutants of green fluorescent protein of Aequora victoria which exhibit environment sensitive excitation and/or emission spectra and are useful, for example, as reporter moieties in the hybrid molecules of this invention. Examples of environment-sensitive GFP mutants provided by this invention are various pH sensitive mutants, which are termed xe2x80x9cpHluorinsxe2x80x9d. Two preferred types of GFP mutants which are provided by this invention are mutants which, in response to a reduction in pH, from pH 7.4 to 5.5 exhibit attenuation or loss of the GFP excitation peak at 475 nm (ecliptic pHluorins) and mutants which exhibit an inverse in the ratio of the excitation peaks at 395 and 475 nm upon a reduction in pH from 7.4 to 6.0 (ratiometric pHluorins). The nucleic acid molecules encoding the amino acid sequences of these GFP mutants are also within the scope of this invention.
In another embodiment of this invention, hybrid reporter molecules which comprise a pHluorin and at least a portion of a vesicle membrane protein are provided. These hybrid reporter molecules are termed xe2x80x9csynaptopHluorinsxe2x80x9d.
In another embodiment of this invention, nucleic acid molecules are provided which encode for the hybrid reporter molecule polypeptides of this invention. Preferably, such nucleic acid molecules comprise a promoter which causes expression of the polypeptide in a specific cell. Also provided are vectors and transformed cells containing the nucleic acid molecules of this invention. Various types of cells may be transformed with the nucleic acids of this invention and include primary cells either in vivo or in vitro, cultured cells including cell lines and cells of transgenic animals. Transgenic animals which express the claimed nucleic acids are thus another embodiment of this invention.
The hybrid molecules and methods of this invention are useful for detecting contact of molecules in an environment with a second environment. This invention is particularly useful for detecting fusion events associated with cell membranes involving endo or exocytosis. In addition, fusion of liposomes containing the hybrid reporter molecules with cells may also be monitored according to this invention. Screening for molecules which alter exocytotic processes, especially in specific cell populations is also made possible by this invention. In addition, the method of this invention provides a means of simultaneously recording the activity of several cells, for example neuronal cells, because one can distinguish spatially, the source of multiple optical signals generated by the cellular release of the peptides provided by this invention.
By providing a means of detecting release of exocytotic vesicles in discrete cell populations this invention provides a means of identifying the contribution of specific proteins or cell processes to exocytosis by measuring such processes in cells which have been altered in some way, for example by the inactivation of certain genes believed to encode for certain proteins involved in such exocytotic processes. Thus, for example, the use of this invention with animals or cells in which certain genes have been xe2x80x9cknocked outxe2x80x9d may provide useful models for providing information regarding exocytosis in various cell types and under various conditions.
Through the use of the synaptopHluorins of this invention, synaptic transmission at individual boutons, as well as secretion in a variety of cell types may be non-invasively imaged by fluorescence microscopy. It is anticipated that the use of pHluorins can be extended to visualize such diverse trafficking processes as endocytosis, receptor activation, and intercompartmental translocation in individual cells or populations of designated cell types, in cultures, tissues, or intact transparent organisms.
The GFP mutants provided by this invention are useful as optical labels. These mutants may be bound to a specific binding molecule which is one member of a ligand binding pair to detect the presence of the other member of the ligand binding pair. These mutants may also be used as reporters of protein expression. Because of their pH sensitivity, these mutants may also be used to detect pH changes in their environment.
An object of this invention is to provide bifunctional polypeptides which may be localized to specific cell types, cell compartments, or cell locations and which participate in the generation of an optical signal upon contact of the polypeptide with the extracellular space.
Another object of this invention is to provide a method for optically detecting the release of the contents of exocytotic vesicles, especially for example, synaptic vesicles.
Another object of this invention is to provide a method of identifying processes and substances which regulate vesicle release.
Yet another object of this invention is to provide mutants of GFP which exhibit excitation and/or emission spectra which are sensitive to changes in the environment.